In the midgestation embryo, blood flow begins after initiation of the heartbeat and subjects vessel walls to viscous friction, pressure, and stretching. These biomechanical forces induce morphological change and activation of differentiation programs not only in endothelial cells but also in hematopoietic cells of the dorsal aorta. The first true hematopoietic stem cells (HSCs) that arise in this region, referred to as the para-aortic splanchnopleura (PSp), are responsible for life-long hematopoiesis of all blood lineages. We have found that fluid frictional force stimulates genetic pathways critical for definitive hematopoiesis and promotes long-term engraftment in adult recipient mice when applied to PSp cells (Nature 2009, 459:1131-1135 and unpublished data). A number of well-characterized pathways are activated by fluid flow in endothelial cells, yet little is known about the signaling pathways that determine hematopoietic fate. The studies proposed herein aim to identify the mechanosensitive genetic signals that are important for hematopoietic specification and expansion. Further, I will test the ability of soluble molecules to mimic the pro-hematopoietic effects of mechanical force. These studies are designed to define the role of biomechanical stress in regulation of hematopoietic potential and promise to inspire innovative approaches for the expansion of transplantable HSCs in culture. Three aims will test the hypothesis that hematopoietic stem cell emergence and expansion is triggered by biomechanically-responsive pathways that can be stimulated by biochemical and pharmacological compounds. Aim 1. Determine the cell surface phenotype(s) of cells that respond to biomechanical forces within the PSp, the embryonic region from which the first definitive HSCs arise. Aim 2. Define and interrogate genetic pathways activated by biomechanical stimulation in hematopoietic precursors from the PSp. Aim 3. Identify pharmacologic compounds and morphogens that promote specification or expansion of HSCs by mimicry of biomechanical forces. Dr. Pamela Wenzel, a postdoctoral research fellow at Children's Hospital Boston (CHB) has outlined a 5- year career plan that will augment and strengthen her background in developmental hematopoiesis and biomechanics. Under the mentorship of Dr. George Daley, a pioneer in the field of stem cell biology, she seeks to identify the genetic mechanisms that sense and respond to biomechanical forces at the earliest stages of definitive hematopoiesis. Dr. Wenzel will be mentored by an Advisory Committee of international leaders in hematopoiesis, biomechanical engineering, and hemodynamics, including Drs. Leonard Zon, Donald Ingber, and Guillermo Garcma-Cardeqa. Finally, the proposed research will be carried out in the Division of Hematology/Oncology at Children's Hospital Boston, the world's largest research institute at a pediatric medical center and the primary pediatric teaching affiliate of Harvard Medical School. PUBLIC HEALTH RELEVANCE: For several decades, the clinical success of hematopoietic stem cell (HSC) transplantation has been limited by the availability and quality of donor-matched sources of marrow, mobilized peripheral blood, and cord blood. This has led to an urgent need for expansion of transplantable patient-specific or universally compatible hematopoietic cells, and yet efforts to expand the HSC supply ex vivo have been largely unsuccessful to date, resulting in poor self-renewal, skewed multi-lineage potential, and low engraftment efficiencies. The identification of biomechanically activated pathways that promote specification and expansion of hematopoietic cells will broaden our understanding of the various types of signals, soluble and mechanical, that define the hematopoietic niche and, moreover, will advance the field toward establishing alternative, high quality sources of hematopoietic cells that can be used for the treatment of hematologic cancers, anemias, and bone marrow failure syndromes.